The CNS is made from cells that divide to form neuroepithelium, which folds into the fluid-filled neural tube. During the onset of neurogenesis, neuroepithelial cells divide asymmetrically in the VZ and SVZ of the anterior and dorsal neural tube to give rise to radial glia, which produces radially migrating newly born neurons of the neocortex. These neurons find their place in the six neocortical layers in an inside out fashion and mature to exhibit various neuronal phenotypes. GABAergic interneurons are generated mainly in subcortical regions and they migrate tangentially to the neocortex. The last steps of neocortical development are synaptogenesis and neuronal network formation, which include making new connections and removing unnecessary ones in activity dependent manner (McConnell, 1988; Marin and Rubenstein, 2003; Guillemot et al., 2006) (Fig. 1).
Figure 1 Neocortex development in mouse. The formation of layers in the embryonic mouse neocortex from embryonic day 11 (E11) to E17 and the structure of adult mouse neocortex. CP, cortical plate; FL, filament layer; IZ, intermediate zone; MZ, marginal zone; PP, preplate; SP, subplate; SVZ, subventricular zone; VZ, ventricular zone; I-VI, cortical layers. Adapted from Molnar et al., 2006 and reprinted by permission from Federation of European Neuroscience Societies and Blackwell Publishers Ltd.
3.1. Corticogenesis
Dividing NSCs in the mammalian neocortical VZ and SVZ, also known as apical and basal NSCs, first give rise to the subplate and cortical layer I. After this, cortical layers are formed in order VI, V, IV, and last II/III. Layer I is the most superficial and the layer VI is the deepest. The mouse is widely used as a model for mammalian neocortex development so only the events during mouse neocortical development will be discussed (Fig. 1 and 2).
Figure 2 The neocortex contains apical and basal progenitor cells, which generate neurons and glia. a In neuroepithelial cells, nucleus migrates from the apical end to basal end of the cell and these cells divide at the apical surface.bIn radial glial cells, basal side of the interkinetic nuclear migration is limited to a boundary between ventricular and subventricular zones. These cells divide at the apical surface. c In basal progenitors, nucleus migrates to basal boundary and the cells divide in that position at the basal end of the ventricular zone or at the subventricular zone. G1, S, G2 and M are phases of the cell cycle. Adapted and reprinted by permission from Macmillan Publishers Ltd: Nature Reviews Molecular Cell Biology, Götz and Huttner, copyright 2005.
At the onset of cortical neurogenesis, NSCs located at the VZ divide symmetrically first and then change to produce more restricted NPCs by asymmetric divisions and give rise to mainly projection neurons and also astrocytes (Davis and Temple, 1994; Williams and Price, 1995; Nieto et al., 2001). A rather complex inhibition of astrocyte differentiation plays a major role in the sequential generation of neurons before the generation of astrocytes in the neocortex (Qian et al., 2000; Morrow et al., 2001; Sun et al., 2001; Fan et al., 2005; He et al., 2005). As afromentioned, proneural genes, which include Ngn1, Ngn2 and Mash1 are crucial for the neuronal differentiation program and mediating neuronal commitment during neocortical neurogenesis (see2.4.1. Neuronal differentiation) (Fode et al., 2000; Nieto et al., 2001).
The regulation of cell cycle progression, cell cycle length and cell cycle exit affects the number of neurons produced during neurogenesis and the neocortical lamination (Polleux et al., 1997; Caviness et al., 2003; Lukaszewicz et al., 2005). Proneural bHLH genes promote cell cycle exit in some areas of the CNS but this has not been shown in the neocortex (Mizuguchi et al., 2001; Lo et al., 2002). Differentiated neocortical neurons are originally generated from either radial glial apical VZ NPCs or basal SVZ NPCs (Haubensak et al., 2004; Miyata et al., 2004; Noctor et al., 2004). Basal NPCs originate from apical NPCs and this process may be mediated by Ngn2 action on the properties of mitotic cells (Miyata et al., 2004). Neocortical neuronal differentiation may involve sequential expression of certain transcription factors including Pax6, Tbr2, NeuroD and Tbr1 in a temporal order although this has not been shown conclusively (Hevner et al., 2001; Englund et al., 2005; Hevner et al., 2006). Neural subtype identity is regulated by proneural bHLH genes, Ngn1 and Ngn2, and homeodomain genes, such as Pax6, in the developing neocortex (Bertrand et al., 2002; Shirasaki and Pfaff, 2002; Lee and Pfaff,
2003). Pax6 directly regulates the transcription of Ngn2 in cortical NPCs (Scardigli et al., 2003). Pax6 and Emx2 are required to promote corticogenesis (Muzio et al., 2002). On the other hand, interactions between Pax6 and homeobox gene Gsh2 contribute to the formation of the pallial-subpallial border (cortical-subcortical) (Toresson et al., 2000).
Pax6 is also required to generate appropriate functional phenotype in the neocortical neurons (Schuurmans et al., 2004; Kroll and O'Leary, 2005). The highest expression levels of Pax6 are found in the NPCs of the ventral neocortex accompanied by region specific expression of sFrp2 and Dbx1 genes (Kim et al., 2001; Yun et al., 2001; Assimacopoulos et al., 2003). Furthermore, Pax6 or nuclear orphan receptor Tlx are essential for inducing expression of sFrp2 in NPCs of ventral neocortex implicating a function for these genes in the patterning of the lateral telencephalon (Stenman et al., 2003). Pax6 expression in the NPCs of the ventral neocortex is required for the development of the claustrum, endopiriform nucleus, piriform cortex, amygdalar lateral nucleus, amygdalar basolateral nucleus, amydalar basomedial nucleus and the nucleus of the lateral olfactory tract (Stoykova et al., 2000; Tole et al., 2005). The radial glia functions contributing to neurogenesis are supported by Pax6 (Heins et al., 2002; Haubst et al., 2004). Cortical layer formation involves a series of complex events (Mallamaci and Stoykova, 2006) and Pax6 participates in these events by mainly specifying later born neurons of upper neocortical identity (Götz et al., 1998; Fukuda et al., 2000). Supporting studies report abnormalities affecting upper but not lower layers of the neocortex in the absence of Pax6 expression (Tarabykin et al., 2001; Noctor et al., 2004; Schuurmans et al., 2004; Zimmer et al., 2004).
SVZ is possibly the origin of upper layer neurons in the primate neocortex (Lukaszewicz et al., 2005) and in mice (Tarabykin et al., 2001). Subventricular tag (Svet1), a marker for both SVZ NPCs and upper cortical layers at perinatal stages, seems to be abolished from these areas in the absence of Pax6, which further supports a role for Pax6 in upper neocortical layer specification (Tarabykin et al., 2001). Other genes expressed in the VZ and in proliferating SVZ cells that migrate to form upper neocortical layers are Cux1 and Cux2, which also seem to specify upper layers in a Pax6 dependent manner (Nieto et al., 2004; Zimmer et al., 2004). It is not known whether Svet1 and Cux genes are expressed intrinsically or extrinsically in these defined populations of neocortex cells. Tlx is also involved in the generation of upper cortical layers and may cooperate with Pax6 (Stenman et al., 2003; Schuurmans et al., 2004).
So far a number of layer specific genes in the neocortex have been identified (Gray et al., 2004; Guillemot et al., 2006). Some of them such as SOX5, Klf6, Zfp312 and NR4A3 are clearly markers for deeper layers and others such as FoxO1, NR4A2 (Nurr1), bHLHb5 and Lmo4 are markers for upper layers. Some of them also exhibit anteroposteriorly and mediolaterally specific or dynamic expression patterns in addition to dorsoventral layer specific patterns. Such genes include Cadherin-6 and ephrin-45, which are expressed in the parietal cortex. On the other hand, Lmo4 is expressed in the frontal cortex and Clim1a is expressed in the occipital cortex. Decreasing mediolateral gradient expression of NR4A3 has been reported in the deeper layers of neocortex. Some layer-specific genes are also essential for the formation of a given layer or layers. In experiments, where layer-specific genes were rendered inactive or functionally blocked, genes such as Brn1, Brn2, were shown to perturb layer II-IV formation, whereas homeobox transcription factor Otx1 affected layer V and Tbr1 caused abnormalities in layers I and VI and the subplate.
3.2. Radial glia
At the onset of neocortical neurogenesis, radial glia forms from neuroepithelial cells upon downregulation of tight junction genes (Aaku-Saraste et al., 1996) and apical to basal separation of certain proteins (Aaku-Saraste et al., 1997). The radial glial cells exhibit a partially neuroepithelial as well as an astroglial phenotype (Campbell and Götz, 2002;
Kriegstein and Götz, 2003). The radial glia as direct followers for neuroephelial cells represents a more restricted form of neocortical NPCs and most of the neocortical neurons and astrocytes are originated from radial glia (Malatesta et al., 2003; Anthony et al., 2004). Radial glia emerges around embryonic day 10 (E10) in the mouse neocortex and is marked by expression of RC1/RC2 and little later by BLBP (Götz and Barde, 2005).
Radial glia retain features from neuroepithelial cells including expression of intermediate-filament protein nestin (Hartfuss et al., 2001), an apical localization of centrosomes and prominin-1 (Weigmann et al., 1997; Chenn et al., 1998), adherence junctions and associated proteins at the apical end of the lateral plasma membrane (Aaku-Saraste et al., 1997; Wodarz and Huttner, 2003), and basal lamina contact (Halfter et al., 2002). In addition, radial glial cells exhibit apical-basal interkinetic nuclear migration, although this is more restricted compared to neuroepithelial cells (Fig. 2). Several astrocytic markers are expressed in radial glia after the onset of neurogenesis including GLAST, BLBP, S100β, GS, TN-C and vimentin (Götz and Barde, 2005; Götz and Huttner, 2005). The radial glia astrocytic structural phetype includes the emergence of the glycogen granules that are storage sites for glycogen in these cells (Gadisseux and Evrard, 1985). Notch signaling through Hes proteins seems to be essential for maintaining radial glia during neurogenesis (Hatakeyama et al., 2004). Radial glia have limited ability to give rise to only a single type of progeny either astrocytes or oligodendrocytes, or neurons (Malatesta et al., 2003;
Anthony et al., 2004). The traditional role for radial glial fibers, reaching from ventricle lumen to pial surface, is to provide a surface on which the newly born neurons can migrate to their destined neocortical layers (Rakic, 1978; Kriegstein et al., 2006). The radial glia seem to be the progeny of neuroepithelial cells with a more restricted potential to generate different kinds of cell types in the neocortex (Götz and Huttner, 2005).
3.3. Gliogenesis
The onset of gliogenesis occurs at time when neocortical NPCs become responsive to BMP, EGF or the cytokines CNTF, LIF and cardiotrophin-1, and glial specific genes such as GFAP and S100β begin to be expressed (Guillemot, 2007). The competence of neocortical NPCs to respond to cytokine signals is achieved by the action of FGF2 and EGF (Lillien and Gulacsi, 2006). Newly born cortical neurons secrete cytokines to promote gliogenesis, which act through the JAK-STAT pathway (Barnabe-Heider et al., 2005). An essential cytokine may be cardiotrophin-1, since other cytokines of the IL-6 family, CNTF and LIF, are only expressed postnatally (Barnabe-Heider et al., 2005; Miller and Gauthier, 2007). In culture, cytokine TGFβ1 signaling through the activation of SMADs appears to be one of the factors that promote gliogenesis by inducing radial glia to produce astrocytes instead of neurons (Stipursky and Gomes, 2007). BMP and Notch signaling favor gliogenesis primarily by blocking the action of proneural genes (Nakashima et al., 2001; Louvi and Artavanis-Tsakonas, 2006). As Notch signaling is required to maintain radial glia, it seems to be equally important in gliogenesis (Taylor et
al., 2007). Notch signaling through DNA binding protein RBP/J seems to be a key element in glial differentiation and it involves the regulation of glial specification gene Sox9 expression (Taylor et al., 2007).
A proportion of the neocortical oligodendrocytes are generated in subcortical regions from which they migrate tangentially into the neocortex in the same manner as GABAergic interneurons and other proportion are generated from cortical NSCs/NPCs (Thomas et al., 2000; He et al., 2001; Tekki-Kessaris et al., 2001; Ross et al., 2003) (Fig.
3).
Figure 3 Induction of neurogenesis and gliogenesis in the embryonic neocortex. Multiple extracellular signals and transcription factors determine whether progenitors adopt neuronal or astroglial fate. Thick black arrows show transcriptional activation and thin grey arrows show non-transcriptional positive interactions. Thick grey lines represent transcriptional repression and thin grey lines represent non-transcriptional negative interactions. BMP, bone morphogenetic protein; C/EBP, CAAT/enhancer-binding protein;
CNTF, ciliary neurotrophic factor; CSL, DNA-bound transcription factor; CT-1, cardiotrophin-1; EGF, epidermal growth factor; E10, embryonic day 10; FGF2, fibroblast growth factor 2; Hes1/5, bHLH transcription factors; ICD, intracellular domain; Id1/3, inhibitors of differentiation 1/3; LIF, leukemia inhibitor factor; N-COR, nuclear receptor co-repressor; PDGF, platelet-derived growth factor; SMAD, mothers against decapentaplegic homologue; STAT, signal transducer and activator of transcription; Wnt, wingless. Reprinted from Progress in Neurobiology, Vol 83, Guillemot, Cell fate specification in the mammalian telencephalon, Pages 37-52, Copyright 2007, with permission from Elsevier.
3.4. Modes of neural migration in the developing telencephalon
In the developing brain, the migration of newborn cells is directed away from the proliferative zones the VZ and the SVZ. Radial migration is the main mechanism that creates the layered structure of excitatory neurons in the neocortex. The migration of interneurons occurs in a largely tangential manner from subpallium to pallium. In the olfactory bulb, new neurons migrate from the SVZ along the rostral migratory stream (RMS).
The migration of pallial neocortical neurons, which occurs in a ventral to dorsal direction from the embryonic VZ towards the pial surface, is termed radial migration (Rakic, 1971; Marin and Rubenstein, 2003). An important scaffold for migrating neurons is provided by radial glial cells, which are also the progenitors for a subset of neocortical neurons (Miyata et al., 2001; Noctor et al., 2001; Noctor et al., 2002). Radial migration of a newborn neocortical neuron involves four stages including initiation of movement, attachment to radial glial fiber, and locomotion with nucleokinesis, detachment, and laminar positioning (Marin and Rubenstein, 2003). During their radial migration newborn neocortical neurons also make retrograde and tangential moves and acquire a transient multipolar phenotype (Tabata and Nakajima, 2003; Noctor et al., 2004).
Tangential migration is a mode of neuronal migration that is not associated with radial glial guidance or direction in the CNS (O'Rourke et al., 1992; Walsh and Cepko, 1992), but cell motility includes the same steps as radial migration (Kriegstein and Noctor, 2004; Marin et al., 2006). Tangentially migrating cells can acquire diverse morphological appearance with short compact processes, long elongated processes, or branched processes (Marin et al., 2006). Tangential migration includes cell motility along the neuronal scaffold, along axons, or dispersed migration from the subpallium to the pallium. First two modes are involved in the migration of olfactory bulb interneurons along the rostral migratory stream and the migration of Gonadotrophin-releasing hormone neurons (Lois and Alvarez-Buylla, 1994; Wray, 2001). The third mode involves migration of interneurons and oligodendrocytes to the neocortical and hippocampal regions during forebrain development (Wichterle et al., 1999; Corbin et al., 2001; Letinic et al., 2002;
Marin and Rubenstein, 2003).
After creating the neuronal variety in the layered neocortex, synaptic connections are established and strenghtened mainly in an activity-dependent fashion. This operation is very complex and new information is altering the overall picture frequently (Muotri and Gage, 2006; Price et al., 2006; Merkle et al., 2007; Toni et al., 2007) and so will not be discussed further.